Altered cortical trigeminal fields excitability by spreading depolarization revealed with in vivo functional ultrasound imaging combined with electrophysiology.

Spreading depolarization (SD), usually termed cortical spreading depression has been proposed as the pathophysiological substrate of migraine aura and as an endogenous trigger of headache pain. The links between neurovascular coupling and cortical craniofacial nociceptive activities modulated by SD were assessed by combining in vivo local field potential (LFPs) recordings in the primary somatosensory cortex (S1) with functional ultrasound (fUS) imaging of S1 and caudal insular (INS) cortices of anesthetized male rats. A single SD wave triggered in the primary visual cortex elicited an ipsilateral, quadriphasic hemodynamic and electrophysiological response in S1 with an early phase consisting of concomitant increases of relative cerebral blood volume (rCBV) and LFPs. A transient hypoperfusion was then correlated with the beginning of the neuronal silence, followed by a strong increase of rCBV while synaptic activities remained inhibited.LFPs and rCBV recovery period was followed by a progressive increase in S1 and INS baseline activities and facilitation of cortical responses evoked by periorbital cutaneous receptive fields stimulation. Sensitization of cortical ophthalmic fields by SD was bilateral, occurred with precise spatiotemporal profiles and was significantly reduced by pre-treatment with a NMDA antagonist. Combined high-resolution assessing of neurovascular coupling and electrophysiological activities has revealed a useful preclinical tool for deciphering central sensitization mechanisms involved in migraine attacks.SIGNIFICANCE STATEMENTA crucial unsolved issue is whether visual aura and migraine headache are parallel or sequential processes. Here we show that a single spreading depolarization (SD) wave triggered from the primary visual cortex is powerful enough to elicit progressive, sustained increases of hemodynamic and sensory responses to percutaneous periorbital noxious stimuli recorded in S1 and Insular ophthalmic fields. Sensitization of cortical ophthalmic fields by SD was bilateral, occurred with precise spatiotemporal profiles and was significantly reduced by pre-treatment with a NMDA antagonist. Combined high-resolution assessing of neurovascular coupling and electrophysiological activities has revealed a useful preclinical tool for deciphering central sensitization mechanisms involved in migraine attacks.

Functional imaging evidence for task-induced deactivation and disconnection of a major default mode network hub in the mouse brain.

The default mode network (DMN) has been defined in functional brain imaging studies as a set of highly connected brain areas, which are active during wakeful rest and inactivated during task-based stimulation. DMN function is characteristically impaired in major neuropsychiatric diseases, emphasizing its interest for translational research. However, in the mouse, a major preclinical rodent model, there is still no functional imaging evidence supporting DMN deactivation and deconnection during high-demanding cognitive/sensory tasks. Here we have developed functional ultrasound (fUS) imaging to properly visualize both activation levels and functional connectivity patterns, in head-restrained awake and behaving mice, and investigated their modulation during a sensory-task, whisker stimulation. We identified reproducible and highly symmetric resting-state networks, with overall connectivity strength directly proportional to the wakefulness level of the animal. We show that unilateral whisker stimulation leads to the expected activation of the contralateral barrel cortex in lightly sedated mice, while interhemispheric inhibition reduces activity in the ipsilateral barrel cortex. Whisker stimulation also leads to elevated bilateral connectivity in the hippocampus. Importantly, in addition to functional changes in these major hubs of tactile information processing, whisker stimulation during genuine awake resting-state periods leads to highly specific reductions both in activation and interhemispheric correlation within the restrosplenial cortex, a major hub of the DMN. These results validate an imaging technique for the study of activation and connectivity in the lightly sedated awake mouse brain and provide evidence supporting an evolutionary preserved function of the DMN, putatively improving translational relevance of preclinical models of neuropsychiatric diseases.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

Pharmaco-fUS: Quantification of pharmacologically-induced dynamic changes in brain perfusion and connectivity by functional ultrasound imaging in awake mice.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.

NanoPaint: A Tool for Rapid and Dynamic Imaging of Membrane Structural Plasticity at the Nanoscale.

Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here, the nano-resolved population dynamics of QDs is exploited to reconstruct the topography and structural changes of the cell membrane surface with high temporal and spatial resolution. For this proof-of-concept study, bright, small, and stable biofunctional QD nanoconstructs are utilized recognizing the endogenous neuronal cannabinoid receptor 1, a highly expressed and fast-diffusing membrane protein, together with a commercial point-localization microscope. Rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allows precise reconstruction of the membrane surface in less than 1 min with a spatial resolution of tens of nanometers. Access of the QD nanoconstructs to the synaptic cleft enables rapid 3D topological reconstruction of the entire presynaptic component. Successful reconstruction of membrane nano-topology and deformation at the second time-scale is also demonstrated for HEK293 cell filopodia and axons. Named "nanoPaint," this super-resolution imaging technique amenable to any endogenous transmembrane target represents a versatile platform to rapidly and accurately reconstruct the cell membrane nano-topography, thereby enabling the study of the rapid dynamic phenomena involved in neuronal membrane plasticity.

Transcranial functional ultrasound imaging of the brain using microbubble-enhanced ultrasensitive Doppler.

Functional ultrasound (fUS) is a novel neuroimaging technique, based on high-sensitivity ultrafast Doppler imaging of cerebral blood volume, capable of measuring brain activation and connectivity in rodents with high spatiotemporal resolution (100µm, 1ms). However, the skull attenuates acoustic waves, so fUS in rats currently requires craniotomy or a thinned-skull window. Here we propose a non-invasive approach by enhancing the fUS signal with a contrast agent, inert gas microbubbles. Plane-wave illumination of the brain at high frame rate (500Hz compounded sequence with three tilted plane waves, PRF=1500Hz with a 128 element 15MHz linear transducer), yields highly-resolved neurovascular maps. We compared fUS imaging performance through the intact skull bone (transcranial fUS) versus a thinned-skull window in the same animal. First, we show that the vascular network of the adult rat brain can be imaged transcranially only after a bolus intravenous injection of microbubbles, which leads to a 9dB gain in the contrast-to-tissue ratio. Next, we demonstrate that functional increase in the blood volume of the primary sensory cortex after targeted electrical-evoked stimulations of the sciatic nerve is observable transcranially in presence of contrast agents, with high reproducibility (Pearson's coefficient ρ=0.7±0.1, p=0.85). Our work demonstrates that the combination of ultrafast Doppler imaging and injection of contrast agent allows non-invasive functional brain imaging through the intact skull bone in rats. These results should ease non-invasive longitudinal studies in rodents and open a promising perspective for the adoption of highly resolved fUS approaches for the adult human brain.

BDNF-dependent plasticity induced by peripheral inflammation in the primary sensory and the cingulate cortex triggers cold allodynia and reveals a major role for endogenous BDNF as a tuner of the affective aspect of pain.

Painful experiences are multilayered, composed of sensory, affective, cognitive and behavioral facets. Whereas it is well accepted that the development of chronic pain is due to maladaptive neuronal changes, the underlying molecular mechanisms, their relationship to the different pain modalities, and indeed the localization of these changes are still unknown. Brain-derived neurotrophic factor (BDNF) is an activity-dependent neuromodulator in the adult brain, which enhances neuronal excitability. In the spinal cord, BDNF underlies the development and maintenance of inflammatory and neuropathic pain. Here, we hypothesized that BDNF could be a trigger of some of these plastic changes. Our results demonstrate that BDNF is upregulated in the anterior cingulate cortex (ACC) and the primary sensory cortex (S1) in rats with inflammatory pain. Injections of recombinant BDNF (into the ACC) or a viral vector synthesizing BDNF (into the ACC or S1) triggered both neuronal hyperexcitability, as shown by elevated long-term potentiation, and sustained pain hypersensitivity. Finally, pharmacological blockade of BDNF-tropomyosin receptor kinase B (TrkB) signaling in the ACC, through local injection of cyclotraxin-B (a novel, highly potent, and selective TrkB antagonist) prevented neuronal hyperexcitability, the emergence of cold hypersensitivity, and passive avoidance behavior. These findings show that BDNF-dependent neuronal plasticity in the ACC, a structure known to be involved in the affective-emotional aspect of pain, is a key mechanism in the development and maintenance of the emotional aspect of chronic pain.

Activation-dependent subcellular distribution patterns of CB1 cannabinoid receptors in the rat forebrain.

Chronic cannabinoid exposure results in tolerance due to region-specific desensitization and down-regulation of CB1 cannabinoid receptors (CB1Rs). For most G-protein-coupled receptors, internalization closely follows rapid desensitization as an important component of long-term down-regulation. However, in vivo patterns of CB1R internalization are not known. Here we investigate the subcellular redistribution of CB1Rs in the rat forebrain following activation by agonist CP55 940 or inhibition by antagonist/inverse agonist AM251. At steady state, CB1Rs are mainly localized to the cell membrane of preterminal axon shafts and, to a lesser degree, to synaptic terminals. A high proportion of CB1Rs is also localized to somatodendritic endosomes. Inhibition of basal activation by acute AM251 administration decreases the number of cell bodies containing CB1R-immunoreactive endosomes, suggesting that CB1Rs are permanently activated and internalized at steady state. On the contrary, acute agonist treatment induces rapid and important increase of endosomal CB1R immunolabeling, likely due to internalization and retrograde transport of axonal CB1Rs. Repeated agonist treatment is necessary to significantly reduce initially high levels of axonal CB1R labeling, in addition to increasing somatodendritic endosomal CB1R labeling in cholecystokinin-positive interneurons. This redistribution displays important region-specific differences; it is most pronounced in the neocortex and hippocampus and absent in basal ganglia.

Presynaptic nanoscale components of retrograde synaptic signaling.

While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell-dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB1Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB1Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ9-tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell-dependent manner.

Axonal targeting of the 5-HT1B serotonin receptor relies on structure-specific constitutive activation.

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.

Local glycolysis fuels actomyosin contraction during axonal retraction.

In response to repulsive cues, axonal growth cones can quickly retract. This requires the prompt activity of contractile actomyosin, which is formed by the non-muscle myosin II (NMII) bound to actin filaments. NMII is a molecular motor that provides the necessary mechanical force at the expense of ATP. Here, we report that this process is energetically coupled to glycolysis and is independent of cellular ATP levels. Induction of axonal retraction requires simultaneous generation of ATP by glycolysis, as shown by chemical inhibition and genetic knock-down of GAPDH. Co-immunoprecipitation and proximal-ligation assay showed that actomyosin associates with ATP-generating glycolytic enzymes and that this association is strongly enhanced during retraction. Using microfluidics, we confirmed that the energetic coupling between glycolysis and actomyosin necessary for axonal retraction is localized to the growth cone and near axonal shaft. These results indicate a tight coupling between on-demand energy production by glycolysis and energy consumption by actomyosin contraction suggesting a function of glycolysis in axonal guidance.

Type-1 cannabinoid receptor signaling in neuronal development.

The type-1 cannabinoid receptor (CB1R) was initially identified as the neuronal target of Δ(9)-tetrahydrocannabinol (THC), the major psychoactive substance of marijuana. This receptor is one of the most abundant G-protein-coupled receptors in the adult brain, the target of endocannabinoid ligands and a well-characterized retrograde synaptic regulator. However, CB1Rs are also highly and often transiently expressed in neuronal populations in the embryonic and early postnatal brain, even before the formation of synapses. This suggests important physiological roles for CB1Rs during neuronal development. Several recent reviews have summarized our knowledge about the role of the endocannabinoid (eCB) system in neurodevelopment and neurotransmission by focusing on the metabolism of endocannabinoid molecules. Here, we review current knowledge about the effects of the modulation of CB1R signaling during the different phases of brain development. More precisely, we focus on reports that directly implicate CB1Rs during progenitor cell migration and differentiation, neurite outgrowth, axonal pathfinding and synaptogenesis. Based on theoretical considerations and on the reviewed experimental data, we propose a new model to explain the diversity of experimental findings on eCB signaling on neurite growth and axonal pathfinding. In our model, cell-autonomus and paracrine eCBs acting on CB1Rs are part of a global inhibitory network of cytoskeletal effectors, which act in concert with positive-feedback local-excitation loops, to ultimately yield highly polarized neurons.

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions.

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

Clathrin-dependent APP endocytosis and Abeta secretion are highly sensitive to the level of plasma membrane cholesterol.

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-beta (Abeta) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to +40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Abeta.

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

Small and stable sulfobetaine zwitterionic quantum dots for functional live-cell imaging.

We have developed a novel surface coating for semiconductor quantum dots (QDs) based on a heterobifunctional ligand that overcomes most of the previous limits of these fluorescent probes in bioimaging applications. Here we show that QDs capped with bidentate zwitterionic dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands are a favorable alternative to polyethylene glycol-coated nanoparticles since they combine small sizes, low nonspecific adsorption, preserved optical properties, and excellent stability over time and a wide range of pH and salinity. Additionally, these QDs can easily be functionalized with biomolecules such as streptavidin (SA) and biotin. We applied streptavidin-functionalized DHLA-SB QDs to track the intracellular recycling of cannabinoid receptor 1 (CB1R) in live cells. These QDs selectively recognized the pool of receptors at the cell surface via SA-biotin interactions with negligible nonspecific adsorption. The QDs retained their optical properties, allowing the internalization of CB1R into endosomes to be followed. Moreover, the cellular activity was apparently unaffected by the probe.

Ultrafast ultrasound imaging pattern analysis reveals distinctive dynamic brain states and potent sub-network alterations in arthritic animals.

Chronic pain pathologies, which are due to maladaptive changes in the peripheral and/or central nervous systems, are debilitating diseases that affect 20% of the European adult population. A better understanding of the mechanisms underlying this pathogenesis would facilitate the identification of novel therapeutic targets. Functional connectivity (FC) extracted from coherent low-frequency hemodynamic fluctuations among cerebral networks has recently brought light on a powerful approach to study large scale brain networks and their disruptions in neurological/psychiatric disorders. Analysis of FC is classically performed on averaged signals over time, but recently, the analysis of the dynamics of FC has also provided new promising information. Keeping in mind the limitations of animal models of persistent pain but also the powerful tool they represent to improve our understanding of the neurobiological basis of chronic pain pathogenicity, this study aimed at defining the alterations in functional connectivity, in a clinically relevant animal model of sustained inflammatory pain (Adjuvant-induced Arthritis) in rats by using functional ultrasound imaging, a neuroimaging technique with a unique spatiotemporal resolution (100 µm and 2 ms) and sensitivity. Our results show profound alterations of FC in arthritic animals, such as a subpart of the somatomotor (SM) network, occurring several weeks after the beginning of the disease. Also, we demonstrate for the first time that dynamic functional connectivity assessed by ultrasound can provide quantitative and robust information on the dynamic pattern that we define as brain states. While the main state consists of an overall synchrony of hemodynamic fluctuations in the SM network, arthritic animal spend statistically more time in two other states, where the fluctuations of the primary sensory cortex of the inflamed hind paws show asynchrony with the rest of the SM network. Finally, correlating FC changes with pain behavior in individual animals suggest links between FC alterations and either the cognitive or the emotional aspects of pain. Our study introduces fUS as a new translational tool for the enhanced understanding of the dynamic pain connectome and brain plasticity in a major preclinical model of chronic pain.

Targeting of the 5-HT1A serotonin receptor to neuronal dendrites is mediated by Yif1B.

The 5-HT(1A) receptor (5-HT(1A)R) is the most extensively characterized serotonin (5-HT) receptor mainly because of its involvement in the mode of action of antidepressants. The 5-HT(1A)R is confined to the somatodendritic domain of central neurons, where it mediates serotonin-evoked hyperpolarization. Our previous studies underlined the role of the short 5-HT(1A)R C-terminal domain in receptor targeting to dendrites. We used this 17 aa region as bait in a yeast two-hybrid screen, and identified, for the first time, an intracellular protein interacting with the 5-HT(1A)R. This protein is homologous to the yeast Yif1p, previously implicated in vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, but not yet characterized in mammals. We confirmed 5-HT(1A)R-Yif1B interaction by glutathione S-transferase pull-down experiments using rat brain extracts and transfected cell lines. Yif1B is highly expressed in the brain, and specifically in raphe 5-HT(1A)R-expressing neurons. Colocalization of Yif1B and 5-HT(1A)R was observed in small vesicles involved in transient intracellular trafficking. Last, inhibition of endogenous expression of Yif1B in primary neuron cultures by small interfering RNA specifically prevented the addressing of 5-HT(1A)R to distal portions of the dendrites, without affecting other receptors, such as sst2A, P2X(2), and 5-HT(3A) receptors. Together, our results provide strong evidence that Yif1B is a member of the ER/Golgi trafficking machinery, which plays a key role in specific targeting of 5-HT(1A)R to the neuronal dendrites. This finding opens up new pathways for the study of 5-HT(1A)R regulation by partner proteins and for the development of novel antidepressant drugs.

Dynamics of somatostatin type 2A receptor cargoes in living hippocampal neurons.

Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.

Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source.

We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing wide-field fluorescence lifetime imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications.